Heparin derivatives having improved anti-Xa specificity

ABSTRACT

Acetyl ester derivatives of heparin are disclosed. These derivatives exhibit improved Anti-Xa activity in relation to their global anticlotting activity.

This invention relates to heparin derivatives possessing increasedanti-Xa/APTT ratios, and more particularly to acetyl ester derivativesof heparin.

The chemical structure of heparin is complex. Heparin is not a singlecompound, but rather is a mixture of compounds. However, heparin iscommonly thought to primarily be a polymeric substance made up oftetrasaccharide repeating units. On the average each tetrasacchariderepeating unit contains approximately 5 free hydroxyl groups and has amolecular weight of approximately 1229. The average molecular weight ofcommercially available heparin varies from about 10,000 to about 18,000.Thus, on the average, commercially available heparin containsapproximately 8 to 15 tetrasaccharide repeating units.

The term heparin is used in the specification and the claims in itsbroadest sense, in order to designate either a commercial heparin ofpharmaceutical grade or a crude heparin such as obtained by extractionfrom biological material, particularly from mammalian tissue, or amucopolysaccharide exhibiting anticoagulant properties that issynthesized from non heparin sources.

Heparin is the most widely used agent for immediate management of mostthromboembolic disorders, particularly, deep vein thrombosis andpulmonary and systemic emboli. An important problem is that the dosagemust be balanced in such a manner that good thrombosis protection isobtained while bleeding complications are avoided. In many instances,bleeding or hemorrhaging has been a major problem, some investigatorsreporting the incidence of hemorrhaging to be as high as 35 percent.

Heparin functions to block the coagulation cascade at various sites byinterfering with a multiplicity of blood factors including factor Xa.Anti-Xa activity which is accompanied by little global anticoagulantactivity is indicative of strong antithrombotic activity while avoidingthe risk of hemorrhage. It should be noted that heparin simultaneouslydepresses a large number of the coagulation factors participating in thecreation and the maintenance of different forms of hypercoagulability.Thus, heparin's activity appears to be global rather than specific.

APTT and USP anticoagulant assays are recognized as measuring globalanticoagulant activity. We prefer to use the APTT assay to measureglobal anticoagulant activity.

Heparin is currently the medication of choice for preventing the risk ofhypercoagulation, for example the appearance of post-operativethrombosis. However excessive amounts of heparin may be at the origin ofserious hemorrhagies. Hence, considerable care must be exercised toutilize the proper amount of heparin to prevent hypercoagulation withoututilizing a sufficient amount to cause hemorhagies. Hence, it isnecessary to keep a constant watch on the patient and adjustments inheparin administration made depending on the results of bloodcoagulation tests which must be administered at regular intervals.

It is an object of this invention to provide medicaments which overcomethe difficulties and constant testing that are associated with heparinadministration. This is accomplished by providing compounds that aremuch more effective than heparin in preventing the risk ofhypercoagulation when compared to the associated side effect of causinghemorrhagies.

U.S. Pat. No. 4,281,108 discloses a process for obtaining low molecularweight heparin comprising acidification of heparin, depolymerization inthe presence of peroxides, and sulfation. The molecular weights of theproducts are 4,000 to 12,000 daltons. An anti-Xa/APTT ratio greater than1 is claimed for the products of this patent.

U.S. Pat. No. 4,303,651 teaches the depolymerization of heparin withnitrous acid or by periodate oxidation to produce low molecular weightheparin fragments exhibiting improved inhibition of activated factor X.Said low molecular weight heparin fragments contain 14-18 sugar units.

U.S. Pat. No. 4,351,938 discloses a process for the preparation ofheparin derivatives exhibiting an improved anti-Xa value. Said heparinderivatives exhibit a molecular weight of 2,000-7,000 daltons (comparedto the molecular weight of commercial heparin of 10,000 to 25,000daltons) and possess analyzable reducing end groups of which themajority are anhydromannose.

U.S. Pat. No. 4,396,762 discloses a heparin product obtained bydegradation of heparin with heparinase from Flavobacterium heparinum(ATCC 13125) or mutants thereof having ability to reduce the coagulationactivity of factor X while not affecting the coagulation activity ofthrombin.

U.S. Pat. No. 4,401,662 discloses oligosaccharides obtainable fromheparin, said oligosaccharides comprising not more than 8 saccharideunits one of which is an N-sulfate-3-O-sulfate-D-glucosamine unit. Theseoligosaccharides may be separated from heparin by means of gelfiltration and exhibit a highly selective activity against activatedfactor X (factor Xa). This results in a strong antithrombotic activitywhile avoiding the risk of hemorrhage for the patient.

U.S. Pat. No. 4,401,758 teaches a process for producing oligosaccharideshaving a highly selective activity against activated factor X (factorXa) of blood i.e. a strong antithrombotic activity while avoiding therisk of hemorrhage. Said process involves depolymerizing heparin andseparating the desired oligosaccharides by contacting with AT III(antithrombin III) followed by a subsequent separation of the desiredoligosaccharides from AT III.

U.S. Pat. No. 4,415,559 discloses an anticoagulant containing heparinhaving low antithrombin III affinity as an effective ingredient andwhich provides a reduced danger of hemorrhage. The low antithrombin IIIaffinity heparin is separated from commercial heparin by affinitychromatography utilizing a gel lattice to which is bonded antithrombinIII. The desired heparin fraction is not absorbed by the lattice-boundantithrombin III gel.

U.S. Pat. No. 4,438,108 describes a mixture of oligo- andpolysaccharides having an improved anti-thrombotic activity vs.hemorrhagic activity as compared to heparin. The product described inthis patent can be liberated from mammalian tissue by autolysis or withthe aid of proteolytic enzymes followed by isolation using organicsolvents, quaternary aliphatic ammonium compounds and/or a basic ionexchanger.

U.S. Pat. No. 4,438,261 discloses chemically partially depolymerizedheparin having a molecular weight of from about 2000 to 7000 daltons andhaving analyzable reducing end groups of which the majority areanhydromannose groups. This product exhibits an improved therapeuticindex which is defined in this patent as the ratio of the anti-Xaactivity to the USP activity.

U.S. Pat. No. 4,474,770 discloses oligosaccharides obtainable fromheparin, said oligosaccharides comprising not more than 8 saccharideunits one of which is an N-sulfate-D-glucosamine unit. Theseoligosaccharides exhibit a high anti-Xa activity relative to heparinwhile the global coagulation activity relative to heparin is very low.Thus, the oligosaccharides are claimed to be advantageously useful forantithrombotic treatment without hemorrhage risks.

U.S. Pat. No. 4,486,420 discloses heparinic mucopolysaccharide fractionswhich have improved antithrombotic activity in vivo (measured in termsof activity of anti-Xa per milligram) compared to heparin and which aremore selective with respect to anti-Xa activity than heparin. Saidfractions have a molecular weight in the range of about 2,000 to 10,000daltons and are insoluble in alcohol.

U.S. Pat. No. 4,500,519 describes a process for producingmucopolysaccharide heparinic fractions having improved anti-Xa activitycompared to heparin. Said fractions are prepared by depolymerizingheparin to a molecular weight range of 2,000 to 8,000 and separatingfractions having selected terminal structures.

U.S. Pat. No. 4,533,549 discloses a derivative of heparin having amolecular weight of from about 2,500 to 4,000 daltons and improvedanti-Xa activity relative to global anticoagulant activity.

U.K. Pat. No. GB 2,002,406B teaches the sulfation of a low molecularweight heparin having a molecular weight of from 2,600 to 5,500. Animproved antithrombotic activity (anti Xa activity) to the anti-bloodclotting activity (KCCT activity) is claimed for the products of thisinvention vs. heparin.

Canadian Pat. No. 1,195,322 discloses a process for obtaining lowmolecular weight heparin comprising the steps of acidifying normalheparin, and depolymerizing in the presence of an oxidizing agent toobtain a low molecular weight heparin product. An anti-Xa/APTT ratio of"almost two" is disclosed.

L. O. Andersson et al. in THROMBOSIS RESEARCH, Vol. 9, 1976 pages575-583 discusses fractions of varying molecular weight isolated fromheparin. The molecular weights of the fractions varied from 5,000 to40,000. Anti-Xa and APTT tests were run on the various fractions. Ingeneral, the data indicated that the lower molecular weight fractionsexhibited higher anti-Xa values in relation to the APTT values.

The prior art substances derived from heparin and having improvedanti-Xa activity in relation to global anticlotting activity have beenobtained by isolating lower molecular weight fractions from heparinand/or depolymerizing heparin. We have been able to produce substanceshaving improved anti-Xa activity in relation to global activity by usingan entirely different and novel approach.

Unexpectedly, it has been observed that acetyl derivatives of heparinexhibit a high anti-Xa (Coatest anti-Xa test kit from KabiVitrum AB,Stockholm, Sweden) activity in relation to APTT (Activated PartialThromboplastine Time see Andersson et al., Thromb. Res. 9, 575 (1976))activity, the latter being a measure of global anticlotting activity.This overcomes the problems associated with prior art methods ofproducing anticoagulants exhibiting high anti-Xa activity in relation toglobal anticoagulant activity.

The preferred method of forming acetyl derivatives of heparin is by thereaction between acetyl chloride and heparin. However, as is obvious toone skilled in the art, there are numerous methods and variations thatmay be employed to for the acetyl derivatives.

Without limitation as to the scope of the invention, it is theorizedthat a preponderance of the ester groups formed by the reaction of anacid chloride with heparin result from the reaction of the free hydroxylgroups of heparin with the acid chloride.

The following examples are given by way of illustration only and are notto be considered as limiting of this invention.

EXAMPLE I

2 grams of heparin (porcine intestinal mucosa) was added to a 250milliliter round bottom flask protected from the atmosphere by a dryingtube. To this was added 24 milliliters of formamide and 24 millilitersof pyridine. The flask was placed in an oil bath maintained at 40° C. 2milliliters of acetyl chloride was added slowly over a 3-4 hour periodwith agitation and agitation continued overnight.

50 milliliters of water was then added with agitation. The contents ofthe flask were then placed in a 2000 molecular weight cutoff dialysisbag (Spectrum Medical Industries, Los Angeles, Calif.). Dialysis wasconducted against a 1% (w/v) sodium chloride solution for 24 hours. Thedialysis against 1% sodium chloride was repeated three times. Dialysiswas then conducted against water for 24 hours. The dialysis againstwater was then repeated three times.

The contents of the dialysis bag were then lyophilized to obtain a dry,white powder.

EXAMPLE II

The product of example I was analyzed for anti-Xa and found to exhibit avalue of 130 units per milligram. The product of example I was analyzedfor APTT and found to exhibit an APTT value of 34.4 units per milligram.The anti-Xa/APTT ratio was thus found to be 3.8. This compares to theknown ratio of 1.0 for heparin.

EXAMPLE III

The infrared spectrum was obtained on the product of example I. Anabsorption peak was observed at 1732 cm⁻¹. This peak is characteristicof an ester group. Said peak was not present in the starting heparin.

EXAMPLE IV

The number of ester groups per tetrasaccharide unit contained in theproduct of example I was measured following the method of S. Hestrin, J.BIOL. CHEM, vol 180, pages 249-261, 1949. Butyryl choline chloride wasused as the ester standard. A theoretical formula weight of 1229 wasused for a tetrasaccharide unit. The results indicated the presence of1.3 acetyl groups per tetrasaccharide unit.

EXAMPLE V

2 grams of heparin (porcine intestinal mucosa) was added to a 250milliliter round bottom flask protected from the atmosphere by a dryingtube. To this was added 24 milliliters of formamide and 24 millilitersof pyridine. The flask was placed in an oil bath maintained at 40° C. 40milliliters of acetyl chloride was added slowly over a 3-4 hour periodwith agitation and agitation continued overnight.

50 milliliters of water was then added with agitation. The contents ofthe flask were then placed in a 2000 molecular weight cutoff dialysisbag (Spectrum Medical Industries, Los Angeles, Calif.). Dialysis wasconducted against a 1% (w/v) sodium chloride solution for 24 hours. Thedialysis against 1% sodium chloride was repeated three times. Dialysiswas then conducted against water for 24 hours. The dialysis againstwater was then repeated three times.

The contents of the dialysis bag were then lyophilized to obtain a dry,white powder.

EXAMPLE VI

The product of example V was analyzed for anti-Xa and found to exhibit avalue of 79 units per milligram. The product of example V was analyzedfor APTT and found to exhibit an APTT value of 6.5 units per milligram.The anti-Xa/APTT ratio was thus found to be 12.2. This compares to theknown ratio of 1.0 for heparin.

EXAMPLE VII

The number of ester groups per tetrasaccharide unit contained in theproduct of example V was measured following the method of S. Hestrin, J.BIOL. CHEM, vol 180, pages 249-261, 1949. Butyryl choline chloride wasused as the ester standard. A theoretical formula weight of 1229 wasused for a tetrasaccharide unit. The results indicated the presence of5.4 acetyl groups per tetrasaccharide unit.

EXAMPLE VIII

The infrared spectrum was obtained on the product of example V. Anabsorption peak was observed at 1740 cm⁻¹. This peak is characteristicof an ester group. Said peak was not present in the starting heparin.

The above description is for the purpose of teaching the person skilledin the art how to practice the present invention. This description isnot intended to detail all of the obvious modifications and variationsof the invention which will become apparent upon reading. However, theapplicants do intend to include all such obvious modifications andvariations within the scope of their invention which is defined by thefollowing claims.

We claim:
 1. An ester of heparin characterized by an anti-Xa/APTT ratiogreater than 1.5.
 2. An ester of claim 1 wherein the ester is preparedby reacting an acid chloride with heparin.
 3. An ester of claim 1wherein more than 50% of the ester groups are aliphatic.
 4. An ester ofclaim 3 containing more than 0.1 ester groups per tetrasaccharide unit.5. An ester of heparin containing more than 0.1 acetyl groups pertgetrasaccharide unit.
 6. An ester of claim 5 wherein the ester isprepared by reacting acetyl chloride with heparin.
 7. An ester ofheparin formed by replacing the free hydroxyl groups of heparin withester groups, said ester characterized by an anti-Xa/APTT ratio greaterthan 1.5.
 8. An ester of claim 7 wherein the ester is prepared byreacting an acid chloride with heparin.
 9. An ester of heparin formed byreplacing the free hydroxyl groups of heparin with ester groups, saidester containing more than 0.1 acetyl groups per tetrasaccharide unit.10. An ester of claim 9 wherein the ester is prepared by reacting acetylchloride with heparin.
 11. An ester of heparin characterized by ananti-Xa/APTT ratio greater than 1.5, wherein the ester is prepared byreacting an acid chloride with heparin, and wherein more than 50% of theester groups are aliphatic.
 12. An ester of heparin characterized by ananti-Xa/APTT ratio greater than 1.5, wherein the ester is prepared byreacting an acid chloride with heparin, wherein more than 50% of theester groups are aliphatic and wherein the ester contains more than 0.1ester groups per tetrasaccharide unit.
 13. An ester of heparin formed byreplacing the free hydroxyl groups of heparin with ester groups, saidester characterized by an anti-Xa/APTT ratio greater than 1.5, whereinthe ester is prepared by reacting an acid chloride with heparin, andwherein more than 50% of the ester groups are aliphatic.
 14. An ester ofheparin formed by replacing the free hydroxyl groups of heparin withester groups, said ester characterized by an anti-Xa/APTT ratio greaterthan 1.5, wherein the ester is prepared by reacting an acid chloridewith heparin, wherein more than 50% of the ester groups are aliphaticand wherein the ester contains more than 0.1 ester groups pertetrasaccharide unit.
 15. An ester of heparin formed by replacing thefree hydroxyl groups of heparin with ester groups, said estercharacterized by an anti-Xa/APTT ratio greater than 1.5, wherein morethan 50% of the ester groups are aliphatic.
 16. An ester of heparinformed by replacing the free hydroxyl groups of heparin with estergroups, said ester characterized by an anti-Xa/APTT ratio greater than1.5, wherein more than 50% of the ester groups are aliphatic and whereinthe ester contains more than 0.1 ester groups per tetrasaccharide unit.17. A pharmaceutically active composition comprising a pharmaceuticallyacceptable carrier or diluent and, as the pharmaceutically activecomponent, a product as claimed in any one of claims 1-16